Background: Vitiligo, an acquired disorder of the skin, is characterized by depigmented macules and patches. Three pathogenic mechanisms that is immunological, neural, and biochemical have been suggested. One of the newer theories in the pathogenesis of vitiligo is the role of oxidative stress.Aims: We compared the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and its cofactor zinc between cases and controls and related it to body surface area (BSA) involved and stability of the disease.Methods: Sixty vitiligo patients and 60 healthy controls were selected, and their serum SOD, MDA, and zinc levels were compared. The cases were further classified on the basis of BSA involved and stability of the disease. Serum SOD, MDA, and zinc levels were then compared within these groups. Serum SOD and MDA levels were measured using enzymatic methods, and serum zinc levels were measured using a commercially available kit.Results: It was observed that SOD and MDA levels were significantly raised in vitiligo cases than in controls. There was no statistically significant difference in serum zinc levels among the cases and controls. Moreover, the values of SOD and MDA were higher in patients with progressive disease than in patients with stable disease.Conclusions: Our observations support the theory of oxidative stress playing role in the autoimmune pathogenesis of vitiligo and SOD and MDA levels may act as biochemical markers for stability in vitiligo.

Vitiligo is an ancient malady.[1],[2] It is an acquired pigmentary disorder of the skin and mucous membranes that is characterized by circumscribed, depigmented macules, and patches. It occurs in about 1–2% of the world population.[2] On the basis of dermatological outpatient records, the incidence of vitiligo is found to be 0.25–2.5% in India.[3] Vitiligo may develop at any age. Onset of vitiligo has been reported from birth to 81 years.[4]

According to the review conducted by the Vitiligo Global Issues Consensus Conference 2011–2012, vitiligo can be classified in the following clinical forms as shown in [Table 1].[5] Several theories have been suggested for the etiology of vitiligo including auto immune hypothesis, neurogenic hypothesis, self-destructive theory of Lerner, convergence/composite theory, genetic hypothesis, etc., but none of them can completely explain the disease. Oxidative stress is one of the newer hypotheses for the etiology of vitiligo. Melanogenesis produces significant amounts of reactive oxygen species (ROS). Certain environmental factors such as ultraviolet radiation, toxins and stress can also induce ROS leading to oxidative stress. The ROS thus produced such as superoxide (O2−), hydrogen peroxide (H2O2), hydroxyl ion (OH−) are involved in cell signaling, melanogenesis and are also used by phagocytes for their bactericidal action.[6] The increased levels of H2O2 may induce fenton type hydroxylation process converting tyrosine present in melanocytes to catechols. These catechols are then oxidized into reactive o-quinones and take part in haptenation process leading to T cell-mediated destruction of melanocytes.[7] ROS can cause oxidative damage to a vast number of biological molecules by causing lipid peroxidation, deoxyribonucleic acid (DNA) modification, and secretion of inflammatory cytokines.[8]

A comprehensive and integrated antioxidant defense mechanism of skin is crucial in protecting it from ROS. One important line of defense is a system of enzymes, which include superoxide dismutase (SOD), glutathione peroxidase, and catalase and all of these decrease the concentration of most harmful oxidants.[9] Various studies have been done in vitiligo patients on the levels of antioxidant enzymes such as SOD, catalase, glutathione peroxidase, and glutathione reductase and products of oxidative damage like malondialdehyde (MDA). To determine the effects of oxidative stress in the pathogenesis of vitiligo, we compared the levels of MDA, SOD and its cofactor zinc between cases and controls and related it to body surface area (BSA) involved and stability of the disease.

Methods

A case control study was undertaken in the Department of Skin, Sexually Transmitted Diseases and Leprosy in collaboration with the Department of Biochemistry, Government Medical College Amritsar, India after approval by the institutional ethical committee. The study was conducted over a period of 2 years. Written informed consent was obtained from each individual selected in the study in her/his vernacular language. On the basis of the previous studies, effective size was calculated for comparing serum MDA, SOD, and zinc levels among cases and controls. Taking alpha error as 0.05, power required 80% sample size, estimated to be around 56 in each group. So we took 60 subjects in each group. Group A (cases) consisted of 60 clinically and histologically diagnosed cases of vitiligo. Group B (controls) had 60 healthy individuals, and without any present or past history of vitiligo they were selected as controls. The subjects with past or present history of any disease, which may affect oxidative stress (like atherosclerosis, coronary artery disease, diabetes mellitus, and smoking), were excluded from the study.

From vitiligo patients and controls, 2 ml of venous blood was withdrawn in a sterile syringe under aseptic conditions by vein puncture, and collected in sterile vial for biochemical analysis. The blood was allowed to stand for half an hour. After the clot formation, the blood sample was centrifuged at 3000 rpm for 10 min and serum thus obtained was analyzed.

Serum MDA was estimated by using the method of Satoh:[10] 1 ml of serum was added to 2 ml of Trichloroacetic acid- Thiobarbituric acid- Hydrochloric acid reagent, mixed and incubated for 15 min in boiling water bath. After cooling, flocculent precipitates were sedimented by centrifugation at 3000 rpm for 10 min. Absorbance of supernatant was measured at 535 nm against the blank.

Serum SOD was analyzed by using the method of Marklund and Marklund modified by Nandi and Chatterjee:[11],[12] For sample: To 2.8 ml of Tris buffer, 0.1 ml of serum was added and mixed. The reaction was started by adding 0.1 ml of adjusted pyrogallol solution (as per control). Reading was taken at 420 nm exactly after 1 min 30 s (B1) and at 3 min 30 s (B2). The absorbance per 2 min (δB) was recorded. For control: 0.1 ml of pyrogallol was added to 2.9 ml of Tris buffer. Calculations were done to estimate serum levels of SOD in U/ml. Serum zinc levels were estimated by standardized kit method Nitro-PAPS.

To prevent selection bias, randomization of cases and blinding of investigators were done. Randomization was done by selecting every 5th, 10th, 15th, 20th, and so on vitiligo patient attending the vitiligo clinic.

Blinding was done as explained below:

Investigator 1 collected blood samples and gave each sample a specific code. Investigator 2, unaware whether the samples given to him were that of cases or of controls, conducted biochemical investigations and handed over the results to investigator 1, who using the specific codes segregated the results of cases and controls.

All the results were recorded in a prestructured proforma. Chi-square test was used to compare distribution of various non-parametric variables between two study groups (like gender, location, etc.). Student’s t test unpaired/analysis of variance (ANOVA) was used to compare mean values between groups. P < 0.05 was taken as statistically significant. All analyses were done using the Statistical Package for the Social Sciences (SPSS) version 17.0.

Results

General observations: No. of subjects, age, family history of vitiligo and history of other autoimmune disorders are given in [Table 2]. Out of 60 cases of vitiligo, a maximum of 43 (71.66%) subjects were of vitiligo vulgaris, followed by 7 (11.67%) of acrofacial and focal vitiligo each, followed by 3 (5%) of segmental vitiligo. Maximum number of cases, that is, 17 were seen in the 11–20 years age group, followed by 15 in >30 years age group, followed by 14 each in ≤10 years and 21–30 years age group.

The cases were classified on the basis of stability of the disease. A total of 30 cases had stable disease and remaining 30 had progressive disease. Stability was defined as “no new lesions and no increase in previous lesions for at least 1 year”. Cases were also classified on the basis of BSA involved. There were 52 cases with <10% BSA involvement and 8 cases with BSA ≥10%.

The demographic profile of vitiligo cases was similar to earlier published studies.[13],[14],[15],[16],[17] Maximum cases 43 (71.66%) were of vitiligo vulgaris, followed by 7 each (11.67%) of acrofacial and focal vitiligo, followed by 3 (5%) of segmental vitiligo. The results are similar to the study of Singh et al.[18] who reported 54.8, 21.5, 18.5, and 14.5% cases of vitiligo vulgaris, acrofacial vitiligo, focal vitiligo, and segmental vitiligo, respectively. The results of various studies done on MDA, SOD, and zinc levels in vitiligo patients are summarized in [Table 4], [Table 5], and [Table 6], respectively. So our study is a repetition of studies on oxidative stress in vitiligo in Indian population.

There have been various studies, which have shown decreased catalase levels in patients of vitiligo.[7],[19] While in our study, we observed increased serum levels of SOD in patients suffering from vitiligo. Functions of SOD and catalase are shown in [Figure 4]. Thus we can say that increased SOD levels and decreased catalase levels may lead to excess of H2O2 in patients of vitiligo.

It has been observed that certain chemical compounds namely mercaptamines and phenolic compounds (o-quinones) can produce a pattern of depigmentation that is similar to vitiligo.[7] The depigmentation can be explained by the proposed process of haptenation. In this process, the preferred target of o-quinones (exogenous or endogenous) is tyrosinase (key enzyme in melanin synthesis). When o-quinone binds to tyrosinase, it is recognized as an antigen by the Langerhans cells, which present it to Major Histocompatibility Complex-1 in the lymph nodes thus stimulating the synthesis of melanocyte specific cytotoxic T cells. These autoreactive T cells then migrate to the lesional site and selectively destroy the melanocytes causing depigmentation and eventually leading to vitiligo.

The increased levels of H2O2 as proposed above may induce fenton type hydroxylation process converting tyrosine present in melanocytes into catechols. These catechols are then oxidized into reactive o-quinones and take part in haptenation process as explained above.

As the serum SOD and MDA levels were higher in cases as compared to controls, there is high oxidative stress in vitiligo patients. Also it was seen that levels of SOD and MDA were higher in patients with progressive disease and those with large BSA involvement. This implies that people with progressive disease and large BSA involvement had high oxidative stress; thus high levels of SOD and MDA in their serum are leading to excess of H2O2 in their skin, causing vitiligo. SOD and MDA levels may be used as biochemical markers of stability of vitiligo as their values correspond with the progression of disease. This may solve the long-known problem of biochemical markers for stability of the disease, which is a cornerstone in the surgical management of vitiligo.

Limitation of our study was that the 8 cases with BSA ≥ 10% had progressive disease, which may act as a confounding factor, and the finding of increased MDA and SOD levels in these cases may be because of the progressive nature of the disease rather than the large BSA involvement. We compared the biochemical results with BSA involved rather than Vitiligo Area Severity Index (VASI), which would have been more accurate.